More than 150 novel variants of HLA class I genes detected in German Stem Cell Donor Registry and UCLA International Cell Exchange samples

HLA ◽  
2018 ◽  
Vol 91 (3) ◽  
pp. 187-194 ◽  
Author(s):  
V. Balz ◽  
S. Krause ◽  
J. Fischer ◽  
J. Enczmann
Keyword(s):  
Stem Cell ◽  
Hla Class I ◽  
Class I ◽  
International Cell ◽  
Cell Donor ◽  
Novel Variants

On the relevance of HLA class I allele level matching for unrelated stem cell donor transplants in Germany

2005 ◽  
Vol 66 (8) ◽  
pp. 21
Author(s):  
Carlheinz R. Müller ◽  
Kaimo Hirv ◽  
Shraga F. Goldmann ◽  
Klaus Schwarz
Keyword(s):  
Stem Cell ◽  
Hla Class I ◽  
Class I ◽  
Cell Donor

Identification of nine new HLA class I alleles in volunteers from the Singapore stem cell donor registries

2006 ◽  
Vol 68 (6) ◽  
pp. 518-520 ◽  
Author(s):  
T. F. Tang ◽  
L. Hou ◽  
B. Tu ◽  
W. Y. K. Hwang ◽  
A. E. J. Yeoh ◽  
...  
Keyword(s):  
Stem Cell ◽  
Hla Class I ◽  
Class I ◽  
Cell Donor

scholarly journals Fine-mapping of HLA class I and class II genes identified two independent novel variants associated with nasopharyngeal carcinoma susceptibility

2018 ◽  
Vol 7 (12) ◽  
pp. 6308-6316 ◽  
Author(s):  
Tong-Min Wang ◽  
Ting Zhou ◽  
Yong-Qiao He ◽  
Wen-Qiong Xue ◽  
Jiang-Bo Zhang ◽  
...  
Keyword(s):  
Nasopharyngeal Carcinoma ◽  
Fine Mapping ◽  
Hla Class I ◽  
Class Ii ◽  
Class I ◽  
Novel Variants

The Activating KIR Genes 2DS1 and 2DS2 Regulate NK Alloreactivity In Vitro.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 927-927
Author(s):  
Joseph H. Chewning ◽  
Charlotte N. Gudme ◽  
Bo Dupont
Keyword(s):  
Stem Cell ◽  
Nk Cells ◽  
Hematopoietic Stem Cell ◽  
Hla Class I ◽  
Class I ◽  
Target Cells ◽  
Ligand Specificity ◽  
Hematopoietic Stem ◽  
Allogeneic Hsct

Abstract The role of Natural Killer (NK) cells in host protection against viral infection and malignant transformation has been well described. NK cells may also lead to a reduction in post-transplant relapse and improved survival in hematopoietic stem cell transplantation (HSCT) for acute myelogenous leukemia (AML). It has been hypothesized that the genotype for the inhibiting killer immunoglobulin-like receptor (KIR) of the hematopoietic stem cell donor in combination with the HLA class I genotype of the recipient could control NK alloreactivity leading to a reduction in post-transplant complications. The KIR gene family encodes however both activating and inhibiting receptors. Here we test the hypothesis that activating KIRs with ligand specificity for HLA class I may contribute to alloreactivity, and potentially could be a genetic factor of significance in allogeneic HSCT. We tested this hypothesis in studies of two pairs of inhibiting and activating KIRs with highly homologous codon sequences in the extracellular domain, namely KIR2DL2/3-KIR2DS2 and KIR2DL1-KIR2DS1. Both the inhibitory 2DL1 and activating 2DS1 have ligand specificity for HLA-Cw group 2, and 2DL2 and 2DL3, have ligand specificity for HLA-Cw group 1, while the activating 2DS2 does not bind in vitro to C1 group. Using an EBV-transformed B-lymphoblastoid cell line (EBV-BLCL) target cell panel homozygous for HLA Class I alleles, we found that NK cells from donors with KIR haplotypes lacking KIR2DS1 or 2DS2 were not cytotoxic to allogeneic EBV-BLCL, independent of the target HLA class I genotype. Polyclonal NK cells obtained from KIR2DS1 positive and C1 group positive donors mediated NK cytotoxicity against C2 positive targets. In contrast, NK cells from KIR2DS1 positive, C2 group homozygous donors displayed minimal cytotoxicity against the C2 group targets (p<0.01). NK clones generated from 2DS1 positive, C2-group negative individuals were cytotoxic to C2-group target cells, while such NK clones could not be obtained from individuals positive for 2DS1 and cognate ligands. Similar findings were made for the relationship between 2DS2, 2DL2/3 and cognate ligand C1 group. Both polyclonal IL-2 propagated NK cells and NK clones from individuals positive for 2DS2 and homozygous for C2 group displayed specific cytotoxicity against C1 positive target cells. The cytotoxicity of 2DS2 positive, C1 group positive NK cells against the C1 positive BLCLs was minimal (p<0.01). These studies demonstrate that 2DS1 and 2DS2 are activating receptors that can induce an alloantigen response. We also present a model for combinations of KIR and HLA genotypes in which the allogeneic function of KIR2DS1 and 2DS2 is consistently seen in donor NK cells. Activating KIR may therefore play a role in allogeneic HSCT, and could contribute to the balance between activating and inhibiting signals for NK cells in HLA-Cw incompatible donor-recipient combinations. Activating KIR interactions with cognate ligand could potentially also play a role in the innate immune response. In the normal host, the increased affinity of the inhibiting KIR isoforms for HLA class I may prevent auto-reactivity, while the activating isoforms may only function in an HLA restricted pattern in context of specific pathogens or transformed cells. It is possible that the low affinity activating KIR may require additional co-stimulating signals that are up-regulated during cellular stress.

Impact of HLA High-Resolution Matching on Outcomes of Myeloablative Unrelated Donor Hematopoietic Stem Cell Transplantation in Chinese Population

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4361-4361
Author(s):  
He Huang ◽  
Yi Luo ◽  
Jimin Shi ◽  
Yamin Tan ◽  
Xiaoyan Han ◽  
...  
Keyword(s):  
Stem Cell ◽  
High Resolution ◽  
Chinese Population ◽  
Hla Class I ◽  
Class Ii ◽  
Hla Class Ii ◽  
Class I ◽  
Unrelated Donor ◽  
Hematopoietic Stem ◽  
The Impact

Abstract Unrelated donor hematopoietic stem cell transplantation (URD-HSCT) is more frequently associated with severe graft-versus-host disease (GVHD) or graft rejection, and the success of URD-HSCT is influenced by the degree of HLA compatibility between the donor and patient. However, HLA mismatched unrelated donors should to be considerable for patients awaiting allogeneic HSCT who lack a suitable related donor or matched unrelated donor. The purpose of the study was to observed the impact of HLA-A, -B, -DRB1/B3 high-resolution matching on outcomes of URD-HSCT in Chinese population. Patients and methods: 182 patients with hematological malignancies received URD-HSCT (bone marrow, n=130; peripheral blood stem cell, n=52) in our center between Nov. 1998 and May. 2008, and donors were from Chinese Marrow Donor Program (Chinese Mainland) and Tzu Chi Stem Cells Center (Chinese Taiwan), and the median age of all patients was 26 years (range 8–52 years). The selection of unrelated donor relied on donor-recipient HLA-A, -B, -DRB1/B3 matching by high-resolution molecular typing by PCR-SSP or PCR-SSO, with 121 cases of HLA 6/6 alleles matched, 51 cases of 1/6 allele mismatched and 10 cases of 2/6 alleles mismatched. The distribution of single HLA class I or class II mismatching was as follows: 37 HLA class I mismatching with 21 HLA-A and 16 HLA-B, and 14 HLA class II mismatching with 12 HLA-DRB1 and 2 HLA-DRB3. All of the patients were received Bu/Cy or Bu/Cy modified myeloablative conditionging regimen. MMF combined with CsA and short course MTX were performed as aGVHD prophylaxis, while other 18 patients received additional anti-CD25 monoclonal antibody to prevent severe aGVHD. Results: After a median follow-up of 14.9 months, 170 patients achieved sustained engraftment with the engraft failure of 6.6%, early treatment-related mortality (TRM) of all patients was 14.4% at 100 days after transplant, and clinical relapse was observed in 8 patients (16.5%). aGVHD developed in 106 (58.2%) patients of all with grade I–II 82 (45.1%) and grade III–IV 24 (13.1%). By Kaplan-Meier method, the accumulative probability of 5-year overall survival (OS) and disease free survival (DFS) of all patients was 51.65±4.15% and 47.38±4.05%, respectively. The incidences of aGVHD was a little higher in HLA 1–2 alleles mismatched group (n=61) compared to HLA matched group (n=121) (67.2% vs 53.7%, p>0.05), and the incidences of grades I–II and III–IV aGVHD in HLA mismatched transplants were 45.9% and 21.3% respectively, while those in HLA matched transplants were 44.6% and 9.1% respectively. Comparing the outcomes between HLA 1–2 alleles mismatched and HLA matched transplants, the engraft failure were 9.8% and 5.0% (P>0.05), and early TRM were 18.0% and 12.4% (P>0.05), respectively. The Kaplan-Meier probability OS at 5 years were 44.31±6.86% and 55.66±5.11% in HLA mismatched and matched group (P>0.05) respectively. In HLA 2 alleles mismatched URD-HSCT, the incidence of engraft failure and aGVHD were 30.0% and 80.0%, and the outcomes were really inferior to HLA matched transplants. The impact of single HLA class I (n=37) or HLA class II mismatched (n=14) on the results of URD-HSCT had been also studied, and incidences of aGVHD in HLA class I or class II mismatched transplants was not significantly different compared with HLA matched transplants. In HLA class I and class II mismatched URD-HSCT, the engraft failure were 5.4% and 7.1% (p>0.05), and early TRM were 13.5% and 35.7% (p>0.05), respectively. The probability OS at 5 years in single HLA class II mismatched transplants was significantly lower compared with HLA matched transplants (23.81±12.94% vs 55.66±5.11%, p<0.01). Conclusion: URD-HSCT could be optimized by comprehensive and precise donor-recipient alleles matching, however, HLA mismatching was associated with the risk of URD-HSCT. Moreover, HLA 2 alleles mismatches of donor-recipient HLA-A, B, DRB high-resolution matching was correlated with an inferior clinical outcome. For patients with high-risk diseases without a suitable matched unrelated donor, alternative methods to URD-HSCT with a single HLA mismatch may permit early treatment before disease progression. In our study, it also demonstrated that HLA class I mismatching was correlated with a high incidence of aGVHD, and HLA class II mismatching was associated with an inferior overall survival in Chinese population, however, larger studies would have to dissect out the magnitude of the risk incurred with specific mismatches more clearly owing to small patient numbers in each group.

Lack of HLA Class I Ligands for Autologous Inhibitory KIR Is Associated with Improved Survival Following Autologous Stem Cell Transplant for Children with Neuroblastoma.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3322-3322
Author(s):  
Jeffrey M Venstrom ◽  
Junting Zheng ◽  
Reenat S Hasan ◽  
Karen E Danis ◽  
Irene Y Cheung ◽  
...  
Keyword(s):  
Overall Survival ◽  
Stem Cell ◽  
High Risk ◽  
Nk Cells ◽  
Progression Free Survival ◽  
Hla Class I ◽  
Class I ◽  
Free Survival ◽  
Hla Ligand ◽  
Myeloablative Chemotherapy

Abstract Background: In hematopoietic stem cell transplantation (HSCT) for hematologic malignancies, natural killer (NK) cells contribute to tumor eradication such that leukemia patients lacking the HLA class I ligand for the donor NK inhibitory killer Ig-like receptors (KIR) have lower relapse rates and longer survival. Since myeloablative chemotherapy followed by autologous HSCT (ASCT) improves survival for children with high risk neuroblastoma (a tumor sensitive to NK killing) we hypothesize that NK cells may be active in this setting and that KIR-HLA combinations where the patient lacks HLA class I ligands for autologous KIR may be associated with improved clinical outcomes. Methods: 155 children with high risk neuroblastoma received myeloablative chemotherapy followed by ASCT between 1992 and 2004. Most patients received anti-GD2 antibody 3F8 and 13-cis-retinoic acid following ASCT. HLA and KIR genotyping was performed. Patients were segregated according to those with or without HLA class I ligand for autologous inhibitory KIR. We examined the 3 inhibitory KIR groups with identified class I ligands: KIR2DL2/2DL3, which recognize HLA-CAsn80(HLA-C1 group), KIR2DL1 recognizing HLA-C Lys80(HLA-C2 group), and KIR3DL1 recognizing HLA-Bw4; as well as 6 activating KIR and 2 KIR haplotype groups. Overall survival and progression-free survival were estimated by Kaplan-Meier method and hazard ratios by Cox regression. No adjustments were made for multiple comparisons. Comparisons of each end point were based on the log-rank statistics. Results: 66% of the 155 children lacked at least 1 HLA ligand for his/ her inhibitory KIR. With median followup of 66.8 months, patients lacking a KIR ligand (n=103) had a 45% lower risk of death compared with patients with all HLA ligands present (n=52) (HR 0.55; 95% CI 0.33–0.90; P=0.015). Similarly, for progression-free survival, the risk of relapse or death was 39% lower for patients lacking an HLA ligand for inhibitory KIR (HR 0.61; 95% CI 0.39–0.97; P=0.035). In particular, patients lacking the HLA-C1 ligand for KIR2DL2/2DL3 experienced an overall survival benefit (HR 0.34; 95% CI 0.11–1.09; P=0.060). Activating KIR and KIR haplotypes were not associated with survival. Conclusion: Among children with high risk neuroblastoma undergoing ASCT, improved overall and progression-free survival is associated with the absence of one or more HLA class I ligands for the patient’s NK cell inhibitory KIR receptor. KIRHLA immunogenetics may therefore be a novel genetic indicator of prognosis for patients undergoing ASCT. Mechanistically, these findings imply that NK tolerance is modified after ASCT, and that KIR-HLA genotypes may also play a role in antibodybased immunotherapy, since most of these patients received 3F8 antibody. These findings require confirmation in a larger prospective study. Figure Figure

scholarly journals Identification of Naturally Presented HLA Ligands of Enriched Leukemic Progenitor Cells for Peptide-Based Immunotherapy in Acute Myeloid Leukemia (AML)

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4046-4046 ◽  
Author(s):  
Annika Nelde ◽  
Heiko Schuster ◽  
Daniel J. Kowalewski ◽  
Lothar Kanz ◽  
Helmut R. Salih ◽  
...  
Keyword(s):  
Stem Cell ◽  
T Cell ◽  
Progenitor Cells ◽  
Hla Class I ◽  
Class Ii ◽  
Hla Class Ii ◽  
Class I ◽  
Isolation And Identification ◽  
Hla Ligands ◽  
Hla Ligandome

Abstract Several studies demonstrated that peptide-based cancer immunotherapy can induce specific immune responses and affect clinical outcome in a variety of different cancer entities. We recently conducted a study, which directly characterized the antigenic landscape of acute myeloid leukemia (AML) by mass spectrometric analysis of naturally presented HLA ligands and identified a panel of AML-specific CD4+ as well as CD8+ T-cell epitopes as suitable targets for T-cell based immunotherapy (Berlin et al. Leukemia 2015). One main reason for the high relapse rates in AML patients after standard polychemotherapy and allogeneic stem cell transplantation is the presence of minimal residual disease (MRD), which is associated with the persistence of leukemic stem cells (LSCs) in the bone marrow of patients. For clinically effective immunotherapy it is therefore indispensable to target the highly chemotherapy resistant LSCs. Here we present a mass spectrometry-based study, which for the first time analyzes the naturally presented HLA ligandome of stem cell enriched (LSCenr) fractions of primary AML samples to identify novel LSC-associated antigens using the approach of direct peptide isolation and identification. The enrichment of LSCs was performed using fluorescence-activated cell sorting of the originally described phenotype of lineage-negative CD34+CD38- cells of PBMCs from eight AML patients. The original stem cell containing population of 1-3% within the PBMCs of most patients was enriched to >90% purity with cell counts of 20-200x106 for the LSCenr fraction per sample. Consistent with our own previous results, all samples showed comparable expression levels of HLA class I molecules on primary leukemia blasts as well as for the LSCenr fractions, with HLA class I molecule counts ranging from 145,000 to 175,000 molecules/cell for the LSCenr fractions. To specifically identify leukemia-associated antigens on LSCenr cells, the HLA ligandome results obtained from the sorted LSCenrfractions were combined with data acquired from AML blasts of 20 AML patients (HLA class I n=19, HLA class II n=20) in previous studies as well as our normal tissue database that comprises 153 HLA class I and 82 HLA class II ligandomes of various healthy tissues (e.g. blood, bone marrow, spleen, kidney, liver, brain, skin, ovary, bowl). We identified more than 14,600 different naturally presented HLA class I ligands representing ̴6,500 source proteins on LSCenr fractions of primary AML samples (n=8) and their autologous blast cells by mass spectrometry. Overlap analysis of the HLA class I ligandomes of LSCenr fractions and autologous AML blasts with the benign peptidome revealed 45.4% (3,132/6,896) and 40.2% (4,922/12,244) of the LSCenr fraction and the autologous AML blast ligandomes to be represented in the benign-associated HLA ligandome, respectively. 79.1% (5,458/6,896) of the mapped LSCenr fraction ligandome was also presented on autologous AML blasts. 1,029 (14.9%) of these identified HLA class I ligands were presented exclusively on LSCenr fractions and not found on autologous AML blasts, previously analyzed AML blasts or any benign tissue. Furthermore, we were able to identify more than 8,000 different naturally presented HLA class II ligands representing ̴1,700 source proteins. Overlap of the HLA class II ligandomes revealed 45.0% (2,800/4,624) and 39.9% (2,706/6,790) of the LSCenr fraction and autologous AML blast ligandomes to be represented in the benign-associated HLA ligands, respectively. The HLA ligandomes of the LSCenr fraction and the autologous AML blasts showed an overlap of 69.7% (3,224/4,624). 941 (11.5%) HLA class II ligands showed exclusive representation in the LSCenr fraction ligandomes and were never identified on AML blast or benign tissue. These LSC-associated peptides represent highly interesting targets for immunotherapeutic approaches in AML patients and will be further evaluated for their potential to elicit a specific T-cell response. Taken together these preliminary results prove the feasibility of our approach to enrich leukemic progenitor cells of primary AML samples for the successful isolation and identification of HLA presented peptides associated with enriched leukemic progenitor cells. Disclosures Schuster: Immatics Biotechnologies GmbH: Employment. Kowalewski:Immatics Biotechnologies GmbH: Employment.

scholarly journals Impact of HLA Molecular Mismatch on Haploidentical Hematopoietic Stem Cell Transplantation: A Center for International Blood and Marrow Transplant Research Study

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3917-3917
Author(s):  
Jun Zou ◽  
Tao Wang ◽  
Yung-Tsi Bolon ◽  
Shahinaz M. Gadalla ◽  
Steven G.E. Marsh ◽  
...  
Keyword(s):  
Stem Cell ◽  
Board Of Directors ◽  
Research Funding ◽  
Hla Class I ◽  
Marrow Transplant ◽  
Class I ◽  
Advisory Committees ◽  
Hematopoietic Stem ◽  
Blood And Marrow Transplant ◽  
Neutrophil Engraftment

Abstract ABSTRACT BACKGROUND The number of haploidentical hematopoietic stem cell transplantations (haplo-HSCT) being performed has substantially increased in recent years. Single-center studies have previously used in silico algorithms to quantitively measure HLA disparity and shown an association of the number of HLA molecular mismatches with relapse protection and/or increased risk of acute graft-versus-host disease (GVHD) in haplo-HSCT. However, inconsistent results from small studies have made it difficult to understand the full clinical impact of molecular mismatch in haplo-HSCT. OBJECTIVE In the current study, we investigated the potential of the HLA class I and II mismatched eplet (ME) score measured by HLAMatchmaker, as well as ME load at a specific locus to predict outcomes in a registry-based cohort of haplo-HSCT recipients. STUDY DESIGN We analyzed data from patients (n= 1,287) who underwent their first haplo-HSCT for acute lymphoblastic leukemia, acute myeloid leukemia, or myelodysplastic syndrome between 2013 and 2017, as reported to the Center for International Blood and Marrow Transplant Research database. ME load at each HLA locus and total Class-I and -II were scored using the HLAMatchmaker module incorporated in HLA Fusion software v4.3, which identifies predicted eplets based on the crystalized HLA molecule models and identifies ME by comparing donor and recipient eplets. RESULTS In the cohort studied, ME scores derived from total HLA Class I or Class II loci or individual HLA loci were not associated with overall survival, disease-free survival, non-relapse mortality, relapse, acute or chronic GVHD (P< .01). An unexpected strong association was identified between total class II ME load in the GVH direction and slower neutrophil engraftment (HR 0.82; 95% CI, 0.75 - 0.91; P < .0001) and platelet engraftment (HR 0.80; 95% CI, 0.72 - 0.88; P < .0001). This was likely attributable to ME load at the HLA-DRB1 locus, which was similarly associated with slower neutrophil engraftment (HR 0.82; 95% CI, 0.73 - 0.92; P = .001) and slower platelet engraftment (HR 0.76; 95% CI, 0.70 - 0.84; P < .0001). Additional analyses suggested that this effect is attributable to matched vs. mismatched in the GVH direction and not to ME load, as there was no dose effect identified. CONCLUSION These findings contradict those of prior relatively small studies reporting that ME load, as quantified by HLAMatchmaker, was associated with haplo-HSCT outcomes. As the study failed to demonstrate the predictive value of ME from HLA molecules for major clinical outcomes, other molecular mismatch algorithms in haplo-HSCT settings should be tested. Disclosures Lee: Pfizer: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; National Marrow Donor Program: Membership on an entity's Board of Directors or advisory committees; Incyte: Research Funding; Janssen: Other; Takeda: Research Funding; Syndax: Research Funding; AstraZeneca: Research Funding; Kadmon: Research Funding; Amgen: Research Funding.

HLA Class I Typing of Platelets by Multi-Colour Flow Cytometry - A New Powerful Tool for Assessing Survival, Activation Status and Activation Capacity of Transfused Platelets Vs. Autologous Platelets and for Determination of Stem Cell Engraftment After Allogeneic Stem Cell Transplantation.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 22-22 ◽  
Author(s):  
Annette Vetlesen ◽  
Pål Andre Holme ◽  
Torstein Lyberg ◽  
Jens Kjeldsen-Kragh
Keyword(s):  
Flow Cytometry ◽  
Stem Cell ◽  
Stem Cell Transplantation ◽  
Allogeneic Stem Cell Transplantation ◽  
Hla Class I ◽  
Class I ◽  
Hla B27 ◽  
Cell Engraftment ◽  
Activation Capacity

Abstract Abstract 22 Studies of in vivo survival of transfused platelets (PLTs) are usually performed by tracing PLTs labelled with radioactive isotopes. The aim of the present work was to develop a flow cytometry-based method without involving manipulation of PLTs before transfusion, where differences in HLA class I molecules between donor and recipient might be used to trace transfused PLTs. Of 14 fluorochrome-conjugated HLA class I monoclonal antibodies (mAbs) from 7 suppliers only 3 (anti-HLA A2, anti-HLA A9 and anti-HLA B27) were found satisfactory for HLA class I typing of PLTs. As earlier studies have claimed the existence of a considerable exchange of HLA class I antigens between plasma and PLTs, a series of experiments were conducted to examine the exchange of HLA A2, A9 and B27 class I antigens during storage for 4 days. Three pairs of HLA +ve and HLA −ve PLT aphaeresis products were collected in autologous plasma for each of the specificities HLA A2, HLA A9 and HLA B27. Cell-free plasma was prepared from an equal number of HLA +ve and HLA −ve whole blood units with the same specificities. Plasma-free PLTs and cell-free plasma were mixed and stored in PLT storage bags at 22°C in the following combinations: HLA +ve PLTs in HLA −ve plasma, HLA +ve PLTs in HLA +ve plasma, HLA −ve PLTs in HLA −ve plasma and HLA −ve PLTs in HLA +ve plasma. Samples from each PLT concentrate-mix were transferred to sterile non-treated culture plates and stored on a flatbed mixer at 37°C in an atmosphere of 5% CO2 in humidified air. HLA class I surface expression was tested daily during storage. The difference in % between pos PLTs in neg plasma and pos PLTs in pos plasma was in the range of 0.8 – 1.2 at 22°C and 0.8 – 3.3 at 37°C for HLA A2; 3.3 – 1.7 at 22°C and 3.5 – 0 at 37°C for HLA A9, and 3.9 – 1.9 at 22°C and 0 – 2.1 at 37°C for HLA B27. Percent-wise difference between neg PLTs in pos plasma and neg PLTs in neg plasma was in the range of 6.9 – 10 at 22°C and 7.7 – 12.5 at 37°C for HLA A2; 4.5 – 0 at 22°C and 13.6 – 6.5 at 37°C for HLA A9, and 5.3 – 5.3 at 22°C and 5.6 – 4.8 at 37°C for HLA B27. These results indicate that the amounts of eluted vs. adsorbed HLA class I antigens are negligible at both 22°C and 37°C. Hence, this method was applied in a clinical setting to study in vivo survival of transfused PLTs. A patient was transfused with PLTs of non-self HLA class I types and tracing and testing of the transfused PLTs were performed by daily measuring of HLA class I surface expression by multi-colour flow cytometry. PLTs were identified by light scatter properties and expression of CD41. The anti-HLA class I mAbs were used to distinguish transfused PLTs from autologous PLTs. The activation status of transfused PLTs was determined by using anti-CD63. PLT activation capacity was further determined by examining the expression of CD63 on different PLT populations before and after stimulation with thrombin receptor agonist peptide (SFLLRN). In a 52 years old patient with AML undergoing allogeneic stem cell transplantation early stem cell engraftment could be detected by following the PLT production (see figure). After the transplantation the number of autologous HLA A2 +ve PLTs decreased gradually. From day 8 till day 10 there was a 50% decrease in total PLT count, while the number of HLA A2 +ve PLTs was low but stable. From day 10 and onwards a gradual increase of HLA A2 +ve PLTs was seen independently of transfusion. These results were interpreted as engraftment from day 8. The standard criteria for stem cell engraftment - a neutrophile count of > 0, 1 × 109/L - occurred 5 days later in this patient. It was also found that the transfused PLTs in the patient's circulation were not activated, evaluated by CD63 expression, and that the residual activation capacity of the transfused PLTs were reduced compared with the capacity of the autologous PLTs (40% up regulation of CD63 vs. 70%; as a comparison there was a 90% up regulation of CD63 in a healthy individual). In conclusion, HLA class I typing by flow cytometry represents a powerful tool for studies of transfused PLTs. The method can 1) be used to study the survival of 3 different populations of transfused PLTs individually, 2) give early evidence of stem cell engraftment after allogeneic stem cell transplantation, 3) be used to assess in vivo activation of transfused PLTs and 4) be used for functional studies of transfused vs. autologous PLTs, which to our knowledge, is the first time this has been possible. Disclosures: No relevant conflicts of interest to declare.

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